In New Zealand, agricultural activity accounts for the majority of greenhouse gas emissions. Therefore, reducing agricultural emissions of greenhouse gases is important for meeting New Zealand's obligations under the Kyoto Protocol. The Protocol requires reduction of greenhouse gases to 1990 levels by the end of the first commitment period (2008-2012). To this end, agricultural sector groups and the New Zealand government established the Pastoral Greenhouse Gas Research Consortium (PGGRC) to identify means for reducing New Zealand's agricultural greenhouse gas emissions.
An important part of the PGGRC's activities has been research into reducing methane emissions from New Zealand's grazing ruminants. Mitigating methane emissions from ruminants is of commercial interest for two reasons. First, failure to meet commitments under the Kyoto Protocol will force the government to purchase carbon credits. This is currently estimated to cost $350 million. Second, methane production results in the loss of 8-12% of the gross energy produced in the rumen. This energy could be used, instead, to improve ruminant productivity.
Methane is produced in the rumen by microbes called methanogens which are part of the phylum Euryarchaeota within the kingdom Archaea. Most methanogens grow on CO2 and H2 as their sole energy source, but some can use acetate or methyl compounds for growth. Several different genera of methanogenic archaea exist in the rumen, but species of the genus Methanobrevibacter, especially M. ruminantium, and M. smithii are thought to be the predominant methanogens in New Zealand ruminants. M. ruminantium is currently the subject of a genome sequencing project funded by the PGGRC. The project is the first genome sequencing of a rumen methanogen and it aims to build a better understanding of the biology of Methanobrevibacter to discover targets for inhibition of methane formation.
Reducing methane production in the rumen requires the inhibition of methanogens or the inactivation of their methanogenesis pathway. A means of inhibiting methane production is to deliver specific inhibitory molecules into methanogen cells. This may be achieved, for example, by coupling inhibitory molecules to cell-permeabilising peptides. In microbial cells, signal peptides mediate the translocation of extracellular proteins from the inside to the outside of the cell and are suitable for the transport of inhibitory molecules. Therefore, it would be useful to identify signal peptides that have the ability to permeabilise methanogen cells and deliver inhibitors.
Signal peptides, or signal sequences, are typically included in precursor proteins secreted from prokaryotic and eukaryotic cells. The signal peptides are part of a cell-permeabilising extension at the N-terminus of the precursor. The primary amino acid sequence of signal peptides is not conserved apart from the cleavage site for signal peptidase (von Heijne, 1985). Yet, signal peptides do share structural similarities. Signal peptides typically include one to five positively charged N-terminal amino acid residues (n-region) followed by 10 to 15 hydrophobic amino acid residues (h-region). A glycine or proline residue is usually located within the hydrophobic domain and a threonine and/or serine residue(s) form a polar domain (c-region) near the cleavage site (Inouye and Halegoua, 1980; Vlasuk et al., 1983, von Heijne, 1985).
A loop model for signal peptide translocation has been proposed (Inouye et al., 1977; Inouye and Halagoua, 1980) whereby the positively charged N-terminus of the signal peptide interacts with the negatively charged inner surface of the cell membrane. The hydrophobic domain is then drawn into the hydrophobic lipid bilayer of the membrane by forming a loop. The loop eventually includes the cleavage site, which is exposed to the signal peptidase for removal of the signal peptide. One of the barriers to inhibiting or limiting methane formation is the ability to deliver inhibitory compounds into methanogen cells. Thus, there is a need to identify signal peptides that are able to attach to cell membranes and to transport molecules across the lipid bilayer, as useful carriers for cell inhibitors.